Dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications

ABSTRACT

The present invention relates to the development of viral vectors expressing different immunogens from the West Nile Encephalitis Virus (WNV) or the Dengue virus which are able to induce protective humoral and cellular immune responses against WNV or Dengue virus infections. More specifically, the present invention relates to three (3) antigens from WNV (the secreted envelope glycoprotein (E), the heterodimer glycoproteins (pre-M-E) and the NSI protein) and from Dengue virus (the secreted envelope glycoprotein (e), the heterodimer glycoproteins (pre-m-e) and the nsl protein) and their use in vaccinal, therapeutic and diagnostic applications.

FIELD OF THE INVENTION

The present invention relates to West-Nile virus (WNV) and/or Denguevirus derived peptides, and more particularly to polypeptides orpolynucleotides derived from WNV and/or Dengue virus polypeptides orpolynucleotides and their use in the preparation of compositions andvaccines. More specifically, the present invention is concerned withcompositions, vaccines and methods for providing an immune responseand/or a protective immunity to animals against a West-Nile virus or aDengue virus and methods for the diagnosis of West-Nile virus or Denguevirus infection.

BACKGROUND OF THE INVENTION

Flaviviridae are arboviruses (arthropod-borne virus) mainly transportedby mosquitoes and blood-sucking ticks. They are small encapsidatedviruses and their genomes consist of infectious single-stranded andlinear RNA of positive polarity. In Man, flaviviruses cause deadlyhemorrhagic fever or meningo-encephalitis. Yellow fever, dengue feverand Japanese encephalitis are the main tropical flaviviroses. Otherimportant human flaviviroses are Saint Louis encephalitis, tick-bornEuropean encephalitis and West Nile fever.

West Nile fever is a zoonosis associated with a flavivirus which wasfirst isolated in Uganda in 1937. Its transmission cycle calls for birdsas the main reservoir and for blood sucking mosquitoes of the Culexgenus as vectors. Migratory viremic birds transport the virus tofar-away regions where they transmit it anew to ornithophile mosquitoesof the Culex genus. Many species of mammals are permissive for the WestNile virus. Horses are particularly sensitive to the disease but do notparticipate in the cycle of transmission. West Nile fever is endemic inAfrica, Asia, Europe and Australia. Phylogenic studies have revealed theexistence of two strains of viruses: viral line 1 has a worldwidedistribution, and viral line 2 is essentially African. Viral line 1 wasresponsible for enzooties in Romania (1996), Russia (1999), Israel(1998-2000) and more recently in North America where the virus had neverbeen detected before 1999. The viral strains isolated during the recentepidemics in Israel and the United-States are more than 99.7% identical.In the Middle-East and North America, where the virus has taken root, animportant bird mortality rate has been observed among infected birds,notably in Corvidae. In North America, over 4000 subjects were infectedwith the West Nile virus, 250 of which died between the months of Augustand December 2002. At the present time, zoonosis is observed in allregions of the United States. At the moment, there exists no humanvaccine or specific therapy against West Nile fever.

In temperate and sub-tropical regions, human infections may occur duringthe fall season. When a subject is bitten by an infected mosquito, theincubation period lasts approximately one week but less than 20% ofpeople infected with the West Nile virus ever go on to clinicalmanifestations. In its benignant form, the viral infection manifestsitself by an undifferentiated febrile state associated with muscularweakness, headaches and abdominal pain. In less than 1% of infectedsubjects, encephalitis or acute aseptic meningitis may occur.Splenomegaly, hepatitis, pancreatitis and myocarditis are also observed.Flask paralyses similar to a poliomyelitic syndrome have recently beenreported, but fatal cases of viral encephalitis (5% of patients havingsevere neurological disorders) mainly concern fragile subjects and theaged. Inter-human transmission of the virus has also recently beenobserved in the United-States in subjects having undergone organtransplants or having been perfused with contaminated blood products.Intra-uterine transmission of the virus has been reported in theUnited-States. The development of a human vaccine against the West Nilefever is a priority in view of the fact that the zoonosis has taken rootin North America and is expected to propagate in the coming months toCentral America, South America and the Caribbean where dengue fever andyellow fever are already rampant.

Therefore, there is a need for West-Nile virus (WNV) and/or Dengue virusderived peptides, and more particularly to polypeptides orpolynucleotides derived from WNV and/or Dengue virus polypeptides orpolynucleotides and their use in the preparation of compositions andvaccines.

The present invention fulfils these needs and also other needs whichwill be apparent to those skilled in the art upon reading the followingspecification.

SUMMARY OF THE INVENTION

The present invention relates to West-Nile virus and/or Dengue virusderived polypeptides.

More specifically, one object of the invention concerns a purifiedpolypeptide wherein it derives from a West-Nile virus antigen or aDengue virus antigen.

Another object of the invention concerns a purified polyclonal ormonoclonal antibody capable of specifically binding to a polypeptide ofthe invention.

Another object of the invention concerns a purified polynucleotidesequence coding for the polypeptide of the invention and its use fordetecting the presence or absence of a West-Nile virus antigen or aDengue virus antigen in a biological sample.

A further object of the invention concerns a recombinant viral vectorwhich is a recombinant virus comprising a polynucleotide sequence of theinvention.

Another object of the invention is a recombinant measles virus capableof expressing a polypeptide of the invention or comprising, in itsgenome, a polynucleotide of the invention.

Yet, another object of the invention relates to a pharmaceuticalcomposition comprising:

-   -   a) at least one component selected from the group consisting of:        -   a polypeptide of the invention or a functional derivative            thereof;        -   an antibody as defined above;        -   an expression vector as defined above;        -   a polynucleotide of the invention or a fragment thereof,        -   a recombinant viral vector of the invention; and        -   a recombinant measles virus of the invention;        -   and    -   b) a pharmaceutically acceptable vehicle or carrier.

Another object of the invention concerns the use of the pharmaceuticalcomposition of the invention, as an anti-West-Nile virus and/or ananti-Dengue virus agent, or for the preparation of an anti-West-Nilevirus and/or an anti-Dengue virus vaccine.

Another object of the invention relates to a host cell incorporating anexpression vector as defined above or a recombinant viral vector asdefined above.

Furthermore, another object of the invention concerns a method ofproducing a recombinant virus for the preparation of an anti-West-Nilevirus vaccine or an anti-Dengue virus vaccine, the method comprising thesteps of:

-   -   a) providing a host cell as defined above;    -   b) placing the host cell from step a) in conditions permitting        the replication of a recombinant virus capable of expressing a        polypeptide of the invention; and    -   c) isolating the recombinant virus produced in step b).

Another object of the invention concerns a West-Nile virusneutralization assay, comprising the steps of:

-   -   a) contacting VERO cells with West-Nile virus and an antibody;    -   b) culturing said VERO cells under conditions which allow for        West-Nile virus replication; and    -   c) measuring reduction of West-Nile virus replication foci on        said VERO cells.

A further object of the invention is to provide a method for treatingand/or preventing a WNV- or Dengue virus-associated disease or infectionin an animal, the method comprising the step of administering to theanimal an effective amount of at least one element selected from thegroup consisting of:

-   -   a polypeptide or a functional derivative thereof as defined        above;    -   an antibody as defined above;    -   an expression vector as defined above;    -   a polynucleotide or a fragment thereof as defined above;    -   a recombinant viral vector as defined above; and    -   a recombinant measles virus as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequence encoding the secretedglycoprotein E from WNV and identified as SEQ ID NO. 1.

FIG. 2 shows the amino acid sequence of the secreted glycoprotein E fromWNV and identified as SEQ ID NO 5.

FIG. 3 shows the nucleic acid sequence encoding the preM plus Eglycoproteins from WNV and identified as SEQ ID NO. 2.

FIG. 4 shows the amino acid sequence of the preM plus E glycoproteinsfrom WNV and identified as SEQ ID NO 6.

FIG. 5 shows the nucleic acid sequence encoding the NS1 protein from WNVand identified as SEQ ID NO. 3.

FIG. 6 shows the amino acid sequence of the NS1 protein from WNV andidentified as SEQ ID NO 7.

FIG. 7 shows the nucleic acid sequence encoding the preM-E gene fromDengue type 1 virus and identified as SEQ ID NO. 4.

FIG. 8 shows the amino acid sequence of the preM-E gene from Dengue type1 virus and identified as SEQ ID NO 8.

FIG. 9 is a schematic map of the pTM-MVSchw recombinant plasmidsaccording to preferred embodiments of the invention.

FIG. 10 shows the expression of sEWNV by MVSchw-sE_(WNV) recombinant MVin Vero cells. (A) Schematic diagram of MV_(Schw)-sE_(WNV) and virusgrowth. The IS-98-ST1 cDNA coding for sE_(WNV) was inserted into theSchwarz MV genome between the BsiW1 and BssHII sites of the ATU atposition 2. The MV genes are indicated: N (nucleoprotein), PVC(phosphoprotein and V, C proteins), M (matrix), F (fusion), H(hemagglutinin), L (polymerase). T7: T7 RNA polymerase promoter; hh:hammerhead ribozyme, T7t: T7 RNA polymerase terminator; δ: hepatitisdelta virus (HDV) ribozyme; ATU: additional transcription unit. (B)Growth curves of MV. Vero cells were infected with MV_(Schw) (open box)or MV_(Schw)-sE_(WNV) (black box) at a multiplicity of infection (m.o.i)of 0.01 TCID₅₀/cell. At various times post-infection, infectious virusparticles were titered as described in the Methods. (C)Immunofluorescence staining of sE_(WNV) glycoprotein in syncitia ofMV_(Schw)-sE_(WNV)-infected Vero cells fixed 36 h post-infection. Cellswere permeabilized (A, B) or not (C, D) with Triton X-100 and thenimmunostained using anti-WNV HMAF. Magnification: ×1000. No positivesignal was observed in MV_(Schw)-infected cells. (D)Radioimmunoprecipitation (RIP) assay showing the release of sE_(WNV)from MV_(Schw)-sE_(WNV)-infected cells. Vero cells were infected withWNV strain IS-98-ST1 (m.o.i of 5) for 24 h, MV_(Schw) (m.o.i. of 0.1),MV_(Schw)-sE_(WNV) (m.o.i. of 0.1) for 40 h, or mock-infected (MI).Radiolabeled supernatants and cell lysates were immunoprecipitated withspecific anti-MV (α-MV) or anti-WNV (α-WNV) polyclonal antibodies. WNV Eglycoprotein (open arrow head) and sE_(WNV) (black arrow head) areshown.

FIG. 11 shows anti-MVSchw-sE_(WNV) antibodies recognizing the WNV Eglycoprotein. Vero cells were infected with WNV strain IS-98-ST1 (WNV)or mock-infected (No virus). Labeled cell lysates wereimmunoprecipitated with pooled immune sera (dilution 1:100) from miceinoculated with WNV, MVSchw, MVSchw-sE_(WNV) as described in the legendto FIG. 10D. Specific anti-lymphochoriomeningitis virus (LCMV)antibodies were used as a negative control. WNV structural glycoproteinsprM and E and non structural proteins NS3, NS5, NS2A and NS2B are shown.p.c., post-challenge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to West-Nile virus (WNV) and/or Denguevirus derived peptides, and more particularly to polypeptides orpolynucleotides derived from WNV and/or Dengue virus polypeptides orpolynucleotides and their use in the preparation of compositions andvaccines. More specifically, the present invention is concerned withcompositions, vaccines and methods for providing an immune responseand/or a protective immunity to animals against a West-Nile virus or aDengue virus and methods for the diagnosis of West-Nile virus or Denguevirus infection.

As used herein, the term “immune response” refers to the T cell responseor the increased serum levels of antibodies to an antigen, or presenceof neutralizing antibodies to an antigen, such as a WNV or a Denguevirus antigen. The term “immune response” is to be understood asincluding a humoral response and/or a cellular response and/or aninflammatory response.

An “antigen” refers to a molecule, such as a protein or a polypeptide,containing one or more epitopes that will stimulate a host's immunesystem to make a humoral and/or cellular antigen-specific response. Theterm is also used interchangeably with “immunogen”.

The term “protection” or “protective immunity” refers herein to theability of the serum antibodies and cellular response induced duringimmunization to protect (partially or totally) against a West-Nile virusor a Dengue virus. Thus, an animal immunized by the compositions orvaccines of the invention will experience limited growth and spread ofan infectious WNV or Dengue virus.

As used herein, the term “animal” refers to any animal that issusceptible to be infected by a West-Nile virus or a Dengue virus. Amongthe animals which are known to be potentially infected by these viruses,there are, but not limited to, humans, birds and horses.

1. Polynucleotides and Polypeptides

In a first embodiment, the present invention concerns a purifiedpolypeptide characterized in that it derives from a West-Nile virusantigen or a Dengue virus antigen or functional derivative thereof. Ascan be appreciated, a protein/peptide is said to “derive” from aprotein/peptide or from a fragment thereof when such protein/peptidecomprises at least one portion, substantially similar in its sequence,to the native protein/peptide or to a fragment thereof.

The West-Nile virus antigen of the present invention is preferablyselected from the group consisting of secreted envelope glycoprotein(E), heterodimer glycoproteins (PreM-E) and NS1 protein. Morespecifically, the secreted envelope glycoprotein (E) comprises thesequence of SEQ ID NO: 5 or a functional derivative thereof, theheterodimer glycoproteins (PreM-E) comprises the sequence of SEQ ID NO:6 or a functional derivative thereof, and the NS1 protein comprises thesequence of SEQ ID NO: 7 or a functional derivative thereof.

The Dengue virus antigen of the invention is preferably selected fromthe group consisting of secreted envelope glycoprotein (E), heterodimerglycoproteins (PreM-E) and NS1 protein. More specifically, theheterodimer glycoproteins (PreM-E) comprises the sequence of SEQ ID NO:8 or a functional derivative thereof.

According to a preferred embodiment, the polypeptide of the presentinvention has an amino acid sequence having at least 80% homology, oreven preferably 85% homology to part or all of SEQ ID NO:1, of SEQ IDNO:2, of SEQ ID NO:3 or of SEQ ID NO:4.

A “functional derivative”, as is generally understood and used herein,refers to a protein/peptide sequence that possesses a functionalbiological activity that is substantially similar to the biologicalactivity of the whole protein/peptide sequence. In other words, itrefers to a polypeptide or fragment(s) thereof that substantially retainthe same biological functions as the polypeptide of SEQ ID Nos: 5 to 8.A functional derivative of a protein/peptide may or may not containpost-translational modifications such as covalently linked carbohydrate,if such modification is not necessary for the performance of a specificfunction. The term “functional derivative” is intended to the“fragments”, “segments”, “variants”, “analogs” or “chemical derivatives”of a protein/peptide. As used herein, a protein/peptide is said to be a“chemical derivative” of another protein/peptide when it containsadditional chemical moieties not normally part of the protein/peptide,said moieties being added by using techniques well known in the art.Such moieties may improve the protein/peptide solubility, absorption,bioavailability, biological half life, and the like. Any undesirabletoxicity and side-effects of the protein/peptide may be attenuated andeven eliminated by using such moieties.

Yet, more preferably, the polypeptide comprises an amino acid sequencesubstantially the same or having 100% identity with SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4.

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or homology for anoptimal alignment. A program like BLASTx will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of identity analysis arecontemplated in the present invention.

As used herein, the term “polypeptide(s)” refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers and to longer chains generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptide(s)” include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. It will be appreciated that thesame type of modification may be present in the same or varying degreeat several sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Modifications can occur anywhere ina polypeptide, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. Modifications include,for example, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation, selenoylation, sulfation andtransfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance: PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman andCompany, New York (1993); Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990); and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

With respect to protein or polypeptide, the term “isolated polypeptide”or “isolated and purified polypeptide” is sometimes used herein. Thisterm refers primarily to a protein produced by expression of an isolatedpolynucleotide molecule contemplated by invention. Alternatively, thisterm may refer to a protein which has been sufficiently separated fromother proteins with which it would naturally be associated, so as toexist in “substantially pure” form.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-99% by weight,the compound of interest.

Purity is measured by methods appropriate for the compound of interest(e.g. chromatographic methods, agarose or polyacrylamide gelelectrophoresis, HPLC analysis, and the like).

In a second embodiment, the present invention concerns a purifiedpolynucleotide encoding a polypeptide of the invention. Therefore, thepolynucleotide of the invention has a nucleic acid sequence which is atleast 65% identical, more particularly 80% identical and even moreparticularly 95% identical to part or all of any one of SEQ ID NO 5 to 8or functional fragments thereof.

A “functional fragment”, as is generally understood and used herein,refers to a nucleic acid sequence that encodes for a functionalbiological activity that is substantially similar to the biologicalactivity of the whole nucleic acid sequence. In other words, it refersto a nucleic acid or fragment(s) thereof that substantially retains thecapacity of encoding for a polypeptide of the invention.

The term “fragment” as used herein refer to a polynucleotide sequence(e.g., cDNA) which is an isolated portion of the subject nucleic acidconstructed artificially (e.g., by chemical synthesis) or by cleaving anatural product into multiple pieces, using restriction endonucleases ormechanical shearing, or a portion of a nucleic acid synthesized by PCR,DNA polymerase or any other polymerizing technique well known in theart, or expressed in a host cell by recombinant nucleic acid technologywell known to one of skill in the art.

With reference to polynucleotides of the invention, the term “isolatedpolynucleotide” is sometimes used. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated polynucleotide” may comprise a DNA molecule inserted intoa vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eucaryote. An “isolated polynucleotidemolecule” may also comprise a cDNA molecule.

Amino acid or nucleotide sequence “identity” and “similarity” aredetermined from an optimal global alignment between the two sequencesbeing compared. An optimal global alignment is achieved using, forexample, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48:443-453). “Identity” means that an amino acid ornucleotide at a particular position in a first polypeptide orpolynucleotide is identical to a corresponding amino acid or nucleotidein a second polypeptide or polynucleotide that is in an optimal globalalignment with the first polypeptide or polynucleotide. In contrast toidentity, “similarity” encompasses amino acids that are conservativesubstitutions. A “conservative” substitution is any substitution thathas a positive score in the blosum62 substitution matrix (Hentikoff andHentikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By thestatement “sequence A is n % similar to sequence B” is meant that n % ofthe positions of an optimal global alignment between sequences A and Bconsists of identical residues or nucleotides and conservativesubstitutions. By the statement “sequence A is n % identical to sequenceB” is meant that n % of the positions of an optimal global alignmentbetween sequences A and B consists of identical residues or nucleotides.

As used herein, the term “polynucleotide(s)” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. This definition includes, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions or single-, double- andtriple-stranded regions, cDNA, single- and double-stranded RNA, and RNAthat is mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded regions, or a mixture of single- anddouble-stranded regions. In addition, “polynucleotide” as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term “polynucleotide(s)” alsoincludes DNAs or RNAs as described above that contain one or moremodified bases. Thus, DNAs or RNAs with backbones modified for stabilityor for other reasons are “polynucleotide(s)” as that term is intendedherein. Moreover, DNAs or RNAs comprising unusual bases, such asinosine, or modified bases, such as tritylated bases, to name just twoexamples, are polynucleotides as the term is used herein. It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. “Polynucleotide(s)” embraces short polynucleotides or fragmentscomprising at least 6 nucleotides often referred to asoligonucleotide(s). The term “polynucleotide(s)” as it is employedherein thus embraces such chemically, enzymatically or metabolicallymodified forms of polynucleotides, as well as the chemical forms of DNAand RNA characteristic of viruses and cells, including, for example,simple and complex cells which exhibits the same biological function asthe polypeptide encoded by any one of SEQ ID NOS. 1 to 4. The term“polynucleotide(s)” also embraces, short nucleotides or fragments, oftenreferred to as “oligonucleotides”, that due to mutagenesis are not 100%identical but nevertheless code for the same amino acid sequence.

2. Vectors and Cells

In a third embodiment, the invention is also directed to a host, such asa genetically modified cell, comprising any of the polynucleotidesequence according to the invention and more preferably, a host capableof expressing the polypeptide encoded by this polynucleotide. Even morepreferably, the present invention is concerned with a host cell thatincorporates an expression vector or a recombinant viral vector asdefined herein below.

The host cell may be any type of cell (a transiently-transfectedmammalian cell line, an isolated primary cell, or insect cell, yeast(Saccharomyces cerevisiae, Ktuyveromyces lactis, Pichia pastoris), plantcell, microorganism, or a bacterium (such as E. coli). The followingbiological deposit relating to MEF/3T3.Tet-Off/prME.WN # h2 cell linecomprising an expression vector encoding for pseudo-particles of WNVstrain IS-98-ST1 composed of prME complexed glycoproteins was registeredat the Collection Nationale des Cultures de Microorganismes (CNCM) underaccession numbers I-3018 on May 2, 2003.

In a fourth embodiment, the invention is further directed to cloning orexpression vector comprising a polynucleotide sequence as defined above.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector.

A number of vectors suitable for stable transfection of cells andbacteria are available to the public (e.g. plasmids, adenoviruses,baculoviruses, yeast baculoviruses, plant viruses, adeno-associatedviruses, retroviruses, Herpes Simplex Viruses, Alphaviruses,Lentiviruses), as are methods for constructing such cell lines. It willbe understood that the present invention encompasses any type of vectorcomprising any of the polynucleotide molecule of the invention.

According to a preferred embodiment, the vector is a recombinant viralvector which is a recombinant virus comprising a polynucleotide sequenceas defined above. Preferably the recombinant virus is a live attenuatedvirus or a defective virus, such as a recombinant virus selected fromthe group consisting of measles virus, hepatitis B virus, humanpapillomavirus, picornaviridae and lentivirus. More preferably, therecombinant virus is a recombinant measles virus, for instance theSchwarz measles virus strain, which is capable of expressing apolypeptide as defined above or comprises, in its genome, apolynucleotide as defined above.

3. Antibodies

In a fifth embodiment, the invention features purified antibodies thatspecifically bind to the isolated or purified polypeptide as definedabove or fragments thereof. The antibodies of the invention may beprepared by a variety of methods using the polypeptides described above.For example, the West-Nile or Dengue virus antigen, or antigenicfragments thereof, may be administered to an animal in order to inducethe production of polyclonal antibodies. Alternatively, antibodies usedas described herein may be monoclonal antibodies, which are preparedusing hybridoma technology (see, e.g., Hammerling of al., In MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, NY, 1981).

As mentioned above, the present invention is preferably directed toantibodies that specifically bind to a West-Nile antigen or a Denguevirus antigen, or fragments thereof. In particular, the inventionfeatures “neutralizing” antibodies. By “neutralizing” antibodies ismeant antibodies that interfere with any of the biological activities ofany of the WNV antigen or Dengue virus antigen. Any standard assay knownto one skilled in the art may be used to assess potentially neutralizingantibodies. Once produced, monoclonal and polyclonal antibodies arepreferably tested for specific WNV or Dengue virus proteins recognitionby Western blot, immunoprecipitation analysis or any other suitablemethod.

Antibodies that recognize WNV or Dengue virus proteins expressing cellsand antibodies that specifically recognize WNV or Dengue virus proteins(or functional fragments thereof), such as those described herein, areconsidered useful to the invention. Such an antibody may be used in anystandard immunodetection method for the detection, quantification, andpurification of WNV or Dengue virus proteins. The antibody may be amonoclonal or a polyclonal antibody and may be modified for diagnosticpurposes. The antibodies of the invention may, for example, be used inan immunoassay to monitor WNV or Dengue virus proteins expressionlevels, to determine the amount of WNV or Dengue virus proteins orfragment thereof in a biological sample and evaluate the presence or notof a WNV or Dengue virus. In addition, the antibodies may be coupled tocompounds for diagnostic and/or therapeutic uses such as gold particles,alkaline phosphatase, peroxidase for imaging and therapy. The antibodiesmay also be labeled (e.g. immunofluorescence) for easier detection.

With respect to antibodies of the invention, the term “specificallybinds to” refers to antibodies that bind with a relatively high affinityto one or more epitopes of a protein of interest, but which do notsubstantially recognize and bind molecules other than the one(s) ofinterest. As used herein, the term “relatively high affinity” means abinding affinity between the antibody and the protein of interest of atleast 10⁶ M⁻¹, and preferably of at least about 10⁷ M⁻¹ and even morepreferably 10⁸ M⁻¹ to 10¹⁰ M⁻¹. Determination of such affinity ispreferably conducted under standard competitive binding immunoassayconditions which is common knowledge to one skilled in the art. As usedherein, “antibody” and “antibodies” include all of the possibilitiesmentioned hereinafter: antibodies or fragments thereof obtained bypurification, proteolytic treatment or by genetic engineering,artificial constructs comprising antibodies or fragments thereof andartificial constructs designed to mimic the binding of antibodies orfragments thereof. Such antibodies are discussed in Colcher at al. (Q JNucl Med 1998; 42: 225-241). They include complete antibodies, F(ab′)₂fragments, Fab fragments, Fv fragments, scFv fragments, other fragments,CDR peptides and mimetics. These can easily be obtained and prepared bythose skilled in the art. For example, enzyme digestion can be used toobtain F(ab′)₂ and Fab fragments by subjecting an IgG molecule to pepsinor papain cleavage respectively. Recombinant antibodies are also coveredby the present invention.

Alternatively, the antibody of the invention may be an antibodyderivative. Such an antibody may comprise an antigen-binding regionlinked or not to a non-immunoglobulin region. The antigen binding regionis an antibody light chain variable domain or heavy chain variabledomain. Typically, the antibody comprises both light and heavy chainvariable domains, that can be inserted in constructs such as singlechain Fv (scFv) fragments, disulfide-stabilized Fv (dsFv) fragments,multimeric scFv fragments, diabodies, minibodies or other related forms(Colcher et al. Q J Nucl Med 1998; 42: 225-241). Such a derivatizedantibody may sometimes be preferable since it is devoid of the Fcportion of the natural antibody that can bind to several effectors ofthe immune system and elicit an immune response when administered to ahuman or an animal. Indeed, derivatized antibody normally do not lead toimmuno-complex disease and complement activation (type IIIhypersensitivity reaction).

Alternatively, a non-immunoglobulin region is fused to theantigen-binding region of the antibody of the invention. Thenon-immunoglobulin region is typically a non-immunoglobulin moiety andmay be an enzyme, a region derived from a protein having known bindingspecificity, a region derived from a protein toxin or indeed from anyprotein expressed by a gene, or a chemical entity showing inhibitory orblocking activity(ies) against WNV or Dengue virus proteins. The tworegions of that modified antibody may be connected via a cleavable or apermanent linker sequence.

Preferably, the antibody of the invention is a human or animalimmunoglobulin such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgDcarrying rat or mouse variable regions (chimeric) or CDRs (humanized or“animalized”). Furthermore, the antibody of the invention may also beconjugated to any suitable carrier known to one skilled in the art inorder to provide, for instance, a specific delivery and prolongedretention of the antibody, either in a targeted local area or for asystemic application.

The term “humanized antibody” refers to an antibody derived from anon-human antibody, typically murine, that retains or substantiallyretains the antigen-binding properties of the parent antibody but whichis less immunogenic in humans. This may be achieved by various methodsincluding (a) grafting only the non-human CDRs onto human framework andconstant regions with or without retention of critical frameworkresidues, or (b) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods are well known to one skilled in the art.

As mentioned above, the antibody of the invention is immunologicallyspecific to the polypeptide of the present invention and immunologicalderivatives thereof. As used herein, the term “immunological derivative”refers to a polypeptide that possesses an immunological activity that issubstantially similar to the immunological activity of the wholepolypeptide, and such immunological activity refers to the capacity ofstimulating the production of antibodies immunologically specific to theWNV or Dengue virus proteins or derivative thereof. The term“immunological derivative” therefore encompass “fragments”, “segments”,“variants”, or “analogs” of a polypeptide.

4. Compositions and Vaccines

The polypeptides of the present invention, the polynucleotides codingthe same, the polyclonal or monoclonal antibodies, the recombinantmeasles virus produced according to the invention, may be used in manyways for the diagnosis, the treatment or the prevention of WNV- orDengue virus-associated diseases or infection.

In a sixth embodiment, the present invention relates to a compositionfor eliciting an immune response or a protective immunity against a WNVor a Dengue virus. According to a related aspect, the present inventionrelates to a vaccine for preventing and/or treating a WNV- or Denguevirus-associated disease or infection. As used herein, the term“treating” refers to a process by which the symptoms of a WNV- or Denguevirus-associated disease or infection are alleviated or completelyeliminated. As used herein, the term “preventing” refers to a process bywhich a WNV- or Dengue virus-associated disease or infection isobstructed or delayed. The composition or the vaccine of the inventioncomprises a polynucleotide, a polypeptide, an expression vector, arecombinant viral vector, a recombinant measles virus and/or an antibodyas defined above and an acceptable carrier.

As used herein, the expression “an acceptable carrier” means a vehiclefor containing the polynucleotide, the polypeptide, the expressionvector, the recombinant viral vector, the recombinant measles virusand/or the antibody of the invention that can be injected into an animalhost without adverse effects. Suitable carriers known in the artinclude, but are not limited to, gold particles, sterile water, saline,glucose, dextrose, or buffered solutions. Carriers may include auxiliaryagents including, but not limited to, diluents, stabilizers (i.e.,sugars and amino acids), preservatives, wetting agents, emulsifyingagents, pH buffering agents, viscosity enhancing additives, colors andthe like.

Further agents can be added to the composition and vaccine of theinvention. For instance, the composition of the invention may alsocomprise agents such as drugs, immunostimulants (such as α-interferon,β-interferon, γ-interferon, granulocyte macrophage colony stimulatorfactor (GM-CSF), macrophage colony stimulator factor (M-CSF),interleukin 2 (IL2), interleukin 12 (IL12), and CpG oligonucleotides),antioxidants, surfactants, flavoring agents, volatile oils, bufferingagents, dispersants, propellants, and preservatives. For preparing suchcompositions, methods well known in the art may be used.

The amount of polynucleotide, polypeptide, expression vector,recombinant viral vector, recombinant measles virus and/or antibodypresent in the compositions or in the vaccines of the present inventionis preferably a therapeutically effective amount. A therapeuticallyeffective amount of the polynucleotide, the polypeptide, the expressionvector, the recombinant viral vector, the recombinant measles virusand/or the antibody of the invention is that amount necessary to allowthe same to perform their immunological role without causing, overlynegative effects in the host to which the composition is administered.The exact amount of polynucleotide, polypeptide, expression vector,recombinant viral vector, recombinant measles virus and/or antibody tobe used and the composition/vaccine to be administered will varyaccording to factors such as the type of condition being treated, themode of administration, as well as the other ingredients in thecomposition.

5. Methods of Use

In a seventh embodiment, the present invention relates to methods fortreating and/or preventing a WNV- or Dengue virus-associated disease orinfection in an animal are provided. The method comprises the step ofadministering to the animal an effective amount of at least one elementselected from the group consisting of:

-   -   a polypeptide of the invention or a functional derivative        thereof;    -   an antibody as defined above;    -   an expression vector as defined above;    -   a polynucleotide of the invention or a fragment thereof,    -   a recombinant viral vector of the invention; and    -   a recombinant measles virus of the invention.

The vaccine, antibody and composition of the invention may be given toan animal through various routes of administration. For instance, thecomposition may be administered in the form of sterile injectablepreparations, such as sterile injectable aqueous or oleaginoussuspensions. These suspensions may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparations may also besterile injectable solutions or suspensions in non-toxicparenterally-acceptable diluents or solvents. They may be givenparenterally, for example intravenously, intramuscularly orsub-cutaneously by injection, by infusion or per os. The vaccine and thecomposition of the invention may also be formulated as creams,ointments, lotions, gels, drops, suppositories, sprays, liquids orpowders for topical administration. They may also be administered intothe airways of a subject by way of a pressurized aerosol dispenser, anasal sprayer, a nebulizer, a metered dose inhaler, a dry powderinhaler, or a capsule. Suitable dosages will vary, depending uponfactors such as the amount of each of the components in the composition,the desired effect (short or long term), the route of administration,the age and the weight of the animal to be treated. Any other methodswell known in the art may be used for administering the vaccine,antibody and the composition of the invention.

The present invention is also directed to a method of producing arecombinant virus for the preparation of an anti-West-Nile virus vaccineor an anti-Dengue virus vaccine, the method comprising the steps of:

-   -   a) providing a host cell as defined above;    -   b) placing the host cell from step a) in conditions permitting        the replication of a recombinant virus capable of expressing a        polypeptide according to the invention; and    -   c) isolating the recombinant virus produced in step b).

In a further embodiment, a West-Nile virus neutralisation assay isprovided. Accordingly, the assay comprises the steps of:

-   -   a) contacting VERO cells with West-Nile virus and an antibody;    -   b) culturing said VERO cells under conditions which allow for        West-Nile virus replication; and    -   c) measuring reduction of West-Nile virus replication foci on        said VERO cells.

EXAMPLES

The present invention will be more readily understood by referring tothe following examples. These examples are illustrative of the widerange of applicability of the present invention and are not intended tolimit its scope. Modifications and variations can be made thereinwithout departing from the spirit and scope of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice for testing of the present invention,the preferred methods and materials are described.

Example 1 Construction of Measles Viruses (MV) Expressing WNV and DEN1Antigens

In order to test their capacity as vaccine candidates against WNVinfection, recombinant Schwarz measles viruses (MV) expressing these WNVand DEN-1 antigens were constructed. The different genes were introducedin an additional transcription unit in the Schwarz MV cDNA that theinventors previously cloned (pTM-MVSchw) (European Patent Application No02291551.6 filed on Jun. 20, 2002). After rescue of the differentrecombinant Schwarz measles viruses expressing the WNV and DEN-1 genes,their capacity to protect mice from a lethal WNV intraperitonealchallenge, and monkeys from Dengue virus infection will be tested.

MV Vector

Mass vaccination with live attenuated vaccines has reduced the incidenceof measles and its complications dramatically since it was introduced inthe 60's. By now, the vaccine has been given to billions of people andis safe and efficacious. It induces a very efficient, life-long CD4, CD8and humoral immunity after a single injection of 104 TCID50. Moreover,it is easy to produce, cheap, and the means to deliver it worldwidealready exist. The safety of this vaccine is due to several factors: i)The stability of the MV genome which explains that reversion topathogenicity has never been observed. ii) The impossibility for the MVgenome to integrate in host chromosomes since viral replication isexclusively cytoplasmic. iii) The production of the vaccine on safeprimary chick embryo fibroblastic cells. Thus, live attenuated MV couldprovide a safe and efficient pediatric vaccination vector.

MV belongs to the genus Morbillivirus in the family Paramyxoviridae. TheEdmonston MV was isolated in 1954 (32), serially passaged on primaryhuman kidney and amnion cells, then adapted to chick embryo fibroblasts(CEF) to produce Edmonston A and B seeds (see (7, 8) for review).Edmonston B was licensed in 1963 as the first MV vaccine. Furtherpassages of Edmonston A and B on CEF produced the more attenuatedSchwarz and Moraten viruses (33) whose sequences have recently beenshown to be identical (34, 35). Being “reactogenic,” Edmonston B vaccinewas abandoned in 1975 and replaced by the Schwarz/Moraten vaccine. Thisis now the most commonly used measles vaccine (7, 8).

In a previous work, the inventors constructed an infectious cDNA from abatch of commercial Schwarz vaccine, a widely used MV vaccine (EuropeanPatent Application No 02291551.6 filed on Jun. 20, 2002). Theextremities of the cDNA were engineered in order to maximize virus yieldduring rescue. A previously described helper cell-based rescue systemwas adapted by co-cultivating transfected cells on primary chick embryofibroblasts, the cells used to produce the Schwarz vaccine. After twopassages the sequence of the rescued virus was identical to that of thecDNA and of the published Schwarz sequence. Two additional transcriptionunits (ATU) were introduced in the cDNA for cloning foreign geneticmaterial. The immunogenicity of rescued virus was studied in micetransgenic for the CD46 MV receptor and in macaques. Antibody titers inanimals inoculated with low doses of the rescued virus were identical tothose obtained with commercial Schwarz MV vaccine. In contrast, theimmunogenicity of a previously described Edmonston strain-derived MVclone was much lower. This new molecular clone allows producing MVvaccine without having to rely on seed stocks. The ATUs, allow producingrecombinant vaccines based on an approved, efficient and worldwide usedvaccine strain.

Example 2 Construction of Schwarz MV-WNV Recombinant Plasmids

1) Secreted Glycoprotein E from WNV

The WNV env gene encoding the secreted form of the protein was generatedby RT-PCR amplification of viral RNA purified from viral particles (WNVIS-98-ST1 strain). The specific sequence was amplified using PfuTurboDNA polymerase (Stratagene) and specific primers that contain uniquesites for subsequent cloning in pTM-MVSchw vector: MV-WNEnv55′-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3′ (SEQ ID NO: 9) (BsiWI siteunderlined) and MV-WNEnv3 5′-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3′ (SEQ IDNO: 10) (BssHII site underlined). A start and a stop codon were added atboth ends of the gene. The whole sequence generated is 1380 nucleotideslong (see FIG. 1), including the start and the stop codons and respectsthe “rule of six”, stipulating that the nucleotides number of MV genomemust be divisible by 6 (28, 29). The Env protein thus generated containsits signal peptide in N-term (18 aa) and no transmembrane region. Thus,It represents amino acids 275-732 in WNV polyprotein and has thesequence shown in FIG. 2.

2) preM Plus E Glycoproteins from WNV

The WNV gene encoding the preM plus E glycoproteins was generated by PCRamplification of plasmid pVL prM-E.55.1 (clone CNCM 1-2732 deposited onOct. 15, 2001). This expression plasmid encodes the pre-M and E proteinsof WNV (IS-98-ST1 strain). The sequence was amplified using PfuTurbo DNApolymerase (Stratagene) and specific primers that contain unique sitesfor subsequent cloning in pTM-MVSchw vector: MV-WNpreME55′-TATCGTACGATGCAAAAGAAAAGAGGAGGAAAG-3′ (SEQ ID NO: 11) (BsiWI siteunderlined) and MV-WNpreME3 5′-ATAGCGCGCTTAAGCGTGCACGTTCACGGAG-3′ (SEQID NO: 12) (BssHII site underlined). A start and a stop codon were addedat both ends of the gene. The whole sequence generated is 2076nucleotides long (see FIG. 3), including the start and the stop codonsand respects the MV “rule of six”. In this construct, the C-terminuspart of the C protein serves as a prM translocation signal. Both preMand E viral glycoproteins are transmembrane glycoproteins type I. It ispresumed that WNV env preME expressing MV will produce and releasemultimeric forms of preM-E heterodimers exhibiting high immunogenicpotential. The construct represents amino acids 302-789 in WNVpolyprotein and has the sequence shown in FIG. 4.

3) NS1 Protein from WNV

The WNV NS1 gene was generated by RT-PCR amplification of viral RNApurified from viral particles (WNV IS-98-ST1 strain). The specificsequence was amplified using PfuTurbo DNA polymerase (Stratagene) andspecific primers: MV-WNNS15 5′-TATCGTACGATGAGGTCCATAGCTCTCACG-3′ (SEQ IDNO: 13) (BsiWI site underlined). and MV-WNNS135′-ATAGCGCGCTCATTAGGTCTTTTCATCATGTCTC-3′ (SEQ ID NO: 14) (BssHII siteunderlined). A start codon was added at the 5′ end and two stop codonsat the 3′ end of the sequence. The whole sequence is 1110 nucleotideslong (see FIG. 5), including the start and the two stop codons, thusrespecting the “rule of six”. The NS1 protein generated contains itssignal peptide sequence in N-term (23 aa). It represents amino acids769-1136 in WNV polyprotein and has the sequence shown in FIG. 6.

4) preM-E Protein from Dengue Type 1 Virus

The Dengue virus gene encoding the preM plus E glycoproteins wasgenerated by PCR amplification of plasmid pVL pIND/[prM+E] (clone 2)(COURAGEOT, M.-P., et al. 2000, A-glucosidase inhibitors reduce denguevirus production by affecting the initial steps of virion morphogenesisin the endoplasmic reticulum. Journal of Virology 74: 564-572). Thisplasmid encodes the pre-M and E glycoproteins of DEN-1 virus (strainFGA/89). The sequence was amplified using PfuTurbo DNA polymerase(Stratagene) and specific primers that contain unique sites forsubsequent cloning in pTM-MVSchw vector: MV-DEN1preME55′-TATCGTACGATGAACAGGAGGAAAAGATCCGTG-3′ (SEQ ID NO: 15) (BsiWI siteunderlined) and MV-DEN1preME3 5′-ATAGCGCGCTTAAACCATGACTCCTAGGTACAG-3′(SEQ ID NO: 16) (BssHII site underlined). A start and a stop codon wereadded at both ends of the gene. The whole sequence generated is 2040nucleotides long (see FIG. 7), including the start and the stop codonsand respects the MV “rule of six”. In this construct, the C-terminuspart of the C protein serves as a preM translocation signal. Both preMand E viral glycoproteins are transmembrane glycoproteins type I. It ispresumed that DEN-1 env expressing MV will produce and releasemultimeric forms of preM-E heterodimers exhibiting high immunogenicpotential. The construct represents amino acids 95-773 in DEN-1polyprotein and has the sequence shown in FIG. 8.

The same immunogens can be prepared by the same way from DEN-2, DEN-3and DEN-4 serotypes.

5) Insertion into MV Schwarz Vector

The different WNV and DEN-1 nucleotidic sequences were cloned inpCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that nomutations were introduced. After BsiWI/BssHII digestion of thepCR2.1-TOPO plasmids, the DNA fragments were cloned in the pTM-MVSchwvector in ATU position 2 giving plasmids: pTM-MVSchw-EnvWNV,pTM-MVSchw-preMEwnv, pTM-MVSchw-NSIWNV and pTM-MVSchw-preMEDEN-1according to FIG. 9.

Example 3 Recovery of Recombinant MVSchw-EnvWNV, MVSchw-preMEwnv andMVSchw-NS1WNV Viruses

To recover recombinant Schwarz viruses from the plasmids, we used thehelper-cell-based rescue system described by Radecke et al. (11) andmodified by Parks et al. (30). Human helper cells stably expressing T7RNA polymerase and measles N and P proteins (293-3-46 cells, a kind giftfrom MA Billeter) were transfected using the calcium phosphate procedurewith pTM-MVSchw-EnvWNV, pTM-MVSchw-preMEwnv or pTM-MVSchw-NSIWNVplasmids (5 μg) and a plasmid expressing the MV polymerase L gene(pEMC-La, 20 ng, a kind gift from MA Billeter). After overnightincubation at 37° C., the transfection medium was replaced by freshmedium and a heat shock was applied (43° C. for two hours) (30). Aftertwo days of incubation at 37° C., transfected cells were transferred ona CEF cells layer and incubated at 32° C. in order to avoid anyadaptation of the Schwarz vaccine that was originally selected on CEFcells and is currently grown on these cells for safety considerations.Infectious virus was easily recovered between 3 and 7 days followingcocultivation. Syncytia appeared occasionally in CEF, but notsystematically. The recombinant viruses were also rescued by the sametechnique after cocultivation of transfected 293-3-46 helper cells at37° C. with primate Vero cells (african green monkey kidney). In thiscase, syncytia appeared systematically in all transfections after 2 daysof coculture. In order to increase the yield of rescue and because theserecombinant viruses will be used in mice experiments, Vero cells wereused as target cells in place of the usual chick embryo fibroblasts(CEF) (European Patent Application No 02291551.6 files on Jun. 20,2002). Recombinant viruses were passaged two times on Vero cells. Theinventors have previously shown that two passages of the Schwarz viruson Vero cells did not change its immunogenic capacities in macaques(European Patent Application No 02291551.6 files on Jun. 20, 2002).

The recombinant viruses were prepared as described above and theexpression of the transgene in infected cells was checked byimmunofluorescence. To detect WNV Envelope glycoproteins expression,immune sera from mice resistant to WNV infection were used(International Patent Application WO 02/081741). To detect NS1 proteinexpression, the inventors used anti-NS1 Monoclonal antibodies(International Patent Application No WO OO/75665).

Example 4 Vaccination Against West-Nile Virus

West Nile disease has recently emerged as an important mosquito-borneflavivirus infection with numerous fatal cases of human encephalitis,thus urging to develop a safe and efficient vaccine. Measles virus (MV)vaccine, a live-attenuated RNA virus, is one of the safest and mosteffective human vaccine developed so far. The Schwarz vaccine strain ofMV can be used as a vector to immunize against heterologous viral,thereby offering a novel and attractive vaccination strategy againstWest Nile virus (WNV). We evaluated the efficacy of a Schwarz measlesvaccine-derived vector expressing the secreted form of the WNV envelopeE glycoprotein in a mouse model. Vaccination induced high titers ofspecific anti-WNV neutralizing antibodies and protection from a lethalWNV challenge. Passive administration with antisera from immunized micealso provided protection, even after challenge with high doses of WNV.Example 4 is the first report that a live-attenuated recombinant measlesvirus provides efficient protective immunity against an heterologousviral disease. The induction of protective immunity shows that liveattenuated-MV expressing the secreted form of the E glycoprotein is aneffective vaccine against West Nile disease.

Materials and Methods

Cells and Virus.

Vero-NK (African green monkey kidney) cells were maintained in DMEMGlutamax (Invitrogen) supplemented with 5% heat-inactivated fetal bovineserum (FBS). Helper 293-3-46 cells used for viral rescue (11) (a kindgift from M. Billeter, Zurich University) were grown in DMEM/10% FBS andsupplemented with 1.2 mg of G 418 per ml. WNV strain IS-98-ST1 (GenBankaccession number AF 481864) was propagated in mosquito Aedespseudoscutellaris AP61 cell monolayers (13). Purification on sucrosegradients, and virus titration on AP61 cells by focus immunodetectionassay (FIA) were performed as previously described (13, 27).

Mouse Antisera to WNV.

Anti-WNV hyperimmune mouse ascitic fluid (HMAF) was obtained by repeatedimmunization of adult mice with WNV strain IS-98-ST1 followed by theinoculation of sarcoma 180. Mouse polyclonal anti-WNV antibodies wereobtained by immunization of adult BALB/c-MBT congenic mice with 10³ FFUof IS-98-ST1 as described previously (13). The WNV-immune serum wascollected one month after priming.

Construction of pTM-MVSchw-sE_(WNV) Plasmid.

The plasmid pTM-MVSchw that contains an infectious MV cDNA correspondingto the anti-genome of the widely used Schwarz/Moraten MV vaccine strainhas been reported elsewhere (10). Additional transcription units wereintroduced into the viral genome to turn it into a vector expressingforeign proteins. To construct pTM-MVSchw-sE_(WNV), genomic RNA of WNVwas extracted from highly purified IS-98-ST1 virions and reversetranscribed using Titan One-Step RT-PCR kit (Roche MolecularBiochemicals) according to the manufacturer's instructions. An RT-PCRfragment encoding the internal E translocation signal (prM-151 toprM-166) followed by the ectodomain and the stem region of the E protein(E-1 to E-441) was generated using the 5′ primer MV-WNEnv55′-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3′ (SEQ ID NO: 9) containing aBsiWI restriction site (underlined) and the 3′ primer MV-WNEnv35′-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3′ (SEQ ID NO: 10) containing a BssHIIrestriction site (underlined). A start and a stop codon were added atboth ends of the gene. The sequence respects the <<rule of six>>,stipulating that the nucleotides number of MV genome must be multiple of6 (28, 29). The PCR product was directly inserted into pCR2.1-TOPOplasmid (TOPO TA cloning kit, Invitrogen) according to themanufacturer's instructions to give TOPO-sE_(WNV). A 1.4-kb fragmentcontaining truncated E protein with translocation signal sequence wasexcised from TOPO-sE_(WNV) using BsiWI and BssHII and then inserted intoBsiWI/BssHII-digested pTM-MVSchw-ATU2 which contains the additionaltranscription unit (ATU) between the P and M genes of Schwarz MV genome(10, 11). The resulting plasmid was designated pTM-MVSchw-sE_(WNV)(named pTM-MVSchw-EnvWVN in the previous Examples). All constructs wereverified by automated sequencing.

Rescue of Recombinant MVSchw-sE_(WNV) Virus from the Cloned cDNA.

Rescue of recombinant Schwarz MV from the plasmid pTM-MVSchw-sE_(WNV)was performed using the helper-cell-based rescue system described byRadecke et al. (11) and modified by Parks et al. (30). Briefly, humanhelper cells stably expressing T7 RNA polymerase and measles N and Pproteins (293-3-46 cells, a kind gift from MA Billeter, ZurichUniversity) were transfected with 5 μg pTM-MVSchw-sE_(WNV) and 0.02 μgpEMC-La expressing the MV polymerase L gene (a kind gift from MABilleter) using the calcium phosphate procedure. After overnightincubation at 37° C., a heat shock was applied for 2 h at 43° C. Aftertwo days of incubation at 37° C., transfected cells were transferredonto a Vero cell monolayer. Vero cells were used as target cells inplace of the usual chick embryo fibroblasts (CEF) in order to increasethe yield of rescued virus. The inventors have previously shown that twopassages of the Schwarz virus on Vero cells did not change itsimmunogenicity in primates (10). Syncytia that appeared after 2-3 daysof coculture were transferred to 35 mm wells of Vero cells, thenexpanded in 75- and then 150-cm² flasks in DMEM/5% FBS. When syncytiareached 80-90% confluence (usually 36-48 h post-infection), the cellswere scraped in a small volume of OptiMEM (Invitrogen) and frozen andthawed once. After low-speed centrifugation to pellet cellular debris,the supernatant, which contained virus, was stored at −80° C. The titersof MVSchw-sE_(WNV) was determined by an endpoint limit dilution assay onVero cells. The 50% tissue culture infectious doses (TCID₅₀) werecalculated using the Kärber method.

Radioimmunoprecipitation Assay.

Vero cells were starved for 1 h with DMEM without methionine andcysteine (ICN Biomedicals) and labeled 3 h with 250 μCi/ml Tran³⁵S-label(ICN Biomedicals). Cells were lysed with RIPA buffer (20 mM TrisCl, pH8.0, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% deoxycholate, 1% TritonX-100) supplemented with a cocktail of protease inhibitors. RIP assaywas performed as previously described (31). Samples were analyzed bySDS-15% PAGE under reducing conditions.

Mice Experiments.

CD46-IFNAR mice were produced as previously described (10). Adult BALB/cmice were purchased from Janvier Laboratories (Le Genest St Isle,France). Mice were housed under specific pathogen-free conditions at thePasteur Institute. Five to 6-week-old CD46-IFNAR mice were i.p.inoculated with 10⁴ or 10⁶ TCID₅₀ of MV. Acute WNV challenge wasperformed by i.p. inoculation of neurovirulent WNV strain IS-98-ST1(i.p.LD₅₀=10) in Dulbecco's modified phosphate saline buffer (DPBS)supplemented with 0.2% bovine serum albumin (BSA) pH 7.5 (Sigma ChemicalCo.). The animals were monitored daily for signs of morbidity andmortality. All experiments are approved and conducted in accordance withthe guidelines of the Office Laboratory Animal Care at PasteurInstitute.

Anti-WN Vaccination Test with Antigenic Boost.

Adult CD46^(+/−) IFN-α/βR^(−/−) mice were vaccinated over a four weekperiod with the MV-WN sE virus at a dose of 10⁴ DCIP50 (which is a doserecommended for humans) and an antigenic boost was provided by purifiedWNV pseudo-particles that were secreted by MEF/3T3.Tet-Off/WN prME # h2cells.

Humoral Response.

To evaluate the specific antibody response in serum, mice were bled viathe periorbital route at different time after inoculation. Detection ofanti-MV antibodies was performed by ELISA (Trinity Biotech, USA) aspreviously described (10). An anti-mouse antibody-HRP conjugate(Amersham) was used as the secondary antibody. The endpoint titer wascalculated as the reciprocal of the last dilution giving a positiveoptical density value. The presence of anti-WNV antibodies was assessedby ELISA as previously described (13). Briefly, microtitration plaqueswere coated with 106 FFU of highly purified WNV strain IS-98-ST1 andthen incubated with mouse sera dilutions. A test serum was consideredpositive if its optical density was twice the optical density of serafrom immunized control mice.

Neutralization Assay.

Anti-WNV neutralizing antibodies were detected by a FRNT test. Sera fromeach mouse group were pooled and heat-inactivated at 56° C. for 30 min.Vero cells were seeded into 12-well plate (1.5×10⁵ cells/well) for 24 h.Mouse serum samples were serially diluted in MEM Glutamax/2% FBS.Dilutions (0.1 ml) were incubated at 37° C. for 2 h and under gentleagitation with an equal volume of WNV strain IS-98-ST1containing ˜100FFU. Remaining infectivity was then assayed on Vero cell monolayeroverlaid with MEM Glutamax/2% FBS containing 0.8% (W/V) carboxy methylcellulose (BDH). After 2 days of incubation at 37° C. with 5% CO₂, FIAwas performed with anti-WNV HMAF as previously described (27). Thehighest serum dilution tested that reduced the number of FFU by at least90% (FRNT₉₀) was considered the end-point titer.

Passive Transfer of Immune Sera.

Pooled immune sera were transferred into 6-week-old female BALB/c miceintraperitoneally. Mice received injection of 0.1 ml of serial dilutionsof pooled serum samples in DPBS/0.2% BSA one day before WNV inoculation.The challenged mice were observed for more than 3 weeks.

Discussion of the Results

Since its introduction into the United States in 1999, West Nile virus(WNV) infection has been recognized as one of the most seriousmosquito-borne disease in the Western Hemisphere, causing severeneurological disease (meningoencephalitis and poliomyelitis-likesyndrome) in humans. (3). Within the last 4 years, WNV had spreadthrough North America, Central America and the Caribbean (1, 2). It ispresumed that it will reach South America in the coming years. Since2002, the US outbreaks were characterized by an apparent increase inhuman disease severity with 13,000 cases and 500 deaths. Althoughmosquito-borne transmission of WNV predominates, WNV is also transmittedby blood transfusion, organ donations and transplacentaly to the fetus(3). Prevention of West Nile encephalitis is a new public healthpriority and it is imperative that a vaccine be developed (3, 4, 5). Novaccine has been approved for human use so far.

Because WNV can be transmitted across species, there is an urgent needto develop preventive strategies for humans. A rational approach shouldbe to confer a long-term immunity in large groups of individuals, and toboost this immunity in case of WNV outbreaks. Measles virus (MV) vaccinecan now be used as a vector to immunize against heterologous viraldiseases, thereby offering a novel and attractive vaccination strategyagainst WNV. We have recently tested this vector against HIV infection(6). MV vaccine, a live-attenuated RNA virus, is one of the safest andmost effective human vaccine developed so far. It induces a veryefficient, life-long immunity after a single or two injections (7, 8).The MV genome is very stable and reversion of vaccine strains topathogenicity has never been observed. The Schwarz MV strain is used intwo widely used measles vaccines, Attenuavax (Merck and Co. Inc., WestPoint, USA) and Rouvax (Aventis Pasteur, Marcy I'Etoile, France), and inthe combined measles, mumps, and rubella vaccine (MMR) (9). We haverecently generated an infectious cDNA for this strain (10) andintroduced additional transcription units (ATU) into it for cloningforeign genes, based on the work of Radecke et al. (11). The vaccinerescued from the molecular clone was as immunogenic as the parentalvaccine in primates and mice susceptible to MV infection. Thus, thisapproved and widely used MV vaccine can be used as a vector to immunizeindividuals simultaneously against measles and other infectiousdiseases.

WNV is a single-stranded RNA virus of the Flaviviridae family, genusflavivirus, within the Japanese encephalitis antigenic complex (2, 3).The virion is composed of three structural proteins, designated C (coreprotein), M (membrane protein) and E (envelope protein). Protein E,which is exposed on the surface of the virion, is responsible for virusattachment and virus-specific membrane fusion. Because the Eglycoprotein can potentially serve as a major protective immunogen for aWNV vaccine (12), the inventors introduced the WNV cDNA encoding thecarboxyl-terminally truncated E glycoprotein lacking thetransmembrane-anchoring region (residues E-1 to E-441, designatedsE_(WNV) hereinafter) of IS-98-ST1 strain (13) into the infectious cDNAfor the Schwarz MV vaccine (10) (FIG. 10A). WNV strain IS-98-ST1 has thesame neuropathologic properties than the new variant designatedIsr98/NY99 that has been responsible for the recent WNV outbreaks inNorth America and Middle East (13). The WNV sequence was introduced inan ATU located between the phosphoprotein (P) and matrix (M) genes inthe MV genome. The recombinant MVSchw-sE_(WNV) virus was produced aftertransfection of the corresponding plasmid into human helper cellsallowing the rescue of negative-stranded RNA paramyxoviruses (11), thenpropagation in Vero cell cultures. The growth of MV_(Schw)-sE_(WNV) inVero cells was only slightly delayed as compared to that of standardSchwarz MV (MV_(Schw)) (FIG. 10B). After 60 h of infection, the yield ofMV_(Schw)-sE_(WNV) was comparable to that of MV_(Schw). The expressionof sE_(WNV) in MV_(Schw)-sE_(WNV)-infected Vero cells was demonstratedby immunofluorescence and radioimmunoprecipitation (RIP) assays (FIG.10C, D). At 40 h post-infection, the cell surface ofMV_(Schw)-sE_(WNV)-induced syncitia was clearly visualized by anti-WNVimmune serum, indicating that sE_(WNV) is transported along thecompartments of the secretory pathway (FIG. 10C). RIP analysis revealedthat anti-WNV antibodies recognized sE_(WNV) that migrated faster thanauthentic E glycoprotein (FIG. 10D). Interestingly, sE_(WNV) wasdetected in the supernatants of MV_(Schw)-sE_(WNV)-infected Vero cellsat 40 h post-infection (FIG. 10D, panel Supernatants/MV_(Schw)-sE_(WNV),lane α-WNV). Thus, MV_(Schw)-sE_(WNV) expresses a recombinant Eglycoprotein which is secreted efficiently. Immunoblots confirmed thatsE_(WNV) accumulated in the culture medium ofMV_(Schw)-sE_(WNV)-infected Vero cells (data not shown).

Genetically modified mice expressing the human CD46 MV receptor andlacking the interferon α/β receptor (6, 14) (CD46^(+/−) IFN-α/β R^(−/−),abbreviated CD46-IFNAR) that are susceptible to MV (14) were used toassess the immune response induced by MV_(Schw)-sE_(WNV). These micedeficient in IFN-α/β response raise cellular and humoral immuneresponses similar to those of competent mice (6, 10, 15, 16). Two groupsof six CD46-IFNAR mice were inoculated intraperitoneally (i.p.) witheither 10⁴ or 10⁶ tissue culture infective doses (TCID₅₀) ofMV_(Schw)-sE_(WNV). Each group was boosted using the same dose 1 monthafter the first immunization. As a control, CD46-IFNAR mice wereimmunized with 10⁶ TCID₅₀ of “empty” MV_(Schw). One month after thefirst immunization, specific anti-MV antibodies were detected in immunesera from mice inoculated with either MV_(Schw) or MV_(Schw)-sE_(WNV)(Table 1). Mice that received either dose of MV_(Schw)-sE_(WNV)displayed specific anti-WNV antibodies at a dilution of 1:3,000. Onemonth after boosting, the titers of anti-WNV antibodies had reached1:30,000 to 1:200,000 (Table 1) and were highly reactive with the WNV Eglycoprotein (FIG. 11). No anti-WNV antibodies were detected in the seraof any control mice (Table 1 and FIG. 11). These results show that oneinjection of MV_(Schw)-sE_(WNV) induces anti-WNV antibodies, and thatboosting one month after priming increases their titers 10 to 60 times.

Anti-WNV neutralizing activity was measured in MV_(Schw)-sE_(WNV)-immunesera using a focus reduction test (FRNT₉₀) (Table 1). As a positivecontrol, the WNV-immune serum from immunized BALB/c-MBT congenic mice(13) gave a FRNT₉₀ titer of 50. The immune sera from CD46-IFNAR miceinoculated with “empty” MV_(Schw) had not detectable neutralizingactivity. Immunized CD46-IFNAR mice which received 10⁴ or 10⁶ TCID₅₀ ofMV_(Schw)-sE_(WNV) raised neutralizing antibodies with similar FRNT₉₀titers, and boosting increased their titers from 10 to 200-300. Thesedata show that mice twice inoculated with the recombinantlive-attenuated MV encoding the secreted form of the IS-98-ST1 Eglycoprotein had high levels of anti-WNV antibody with neutralizingactivity, regardless of the injected dose.

Because antibody-mediated immunity may be critical to protect againstWNV infection (17, 18), the inventors examined if the passive transferof sera from MV_(Schw)-sE_(WNV)-immunized mice can protect adult BALB/cmice from WNV infection (Table 2). Groups of six 6-week-old BALB/c micereceived i.p. various amounts of pooled immune sera fromMV_(Schw)-sE_(WNV)-immunized CD46-IFNAR mice collected one month afterpriming or boosting. One day later, the mice were challenged with 10times the i.p. 50% lethal dose (LD₅₀) of WNV strain IS-98-ST1 (13, 19).As a positive control, BALB/c mice that received as little as 2 μl ofthe WNV-immune serum were protected from the challenge (Table 2). Incontrast, all mice that received 2 μl of the non-immune mouse serum orserum from “empty” MV_(Schw)-immunized mice died within 11-12 days.Protective passive immunity was observed in all BALB/c mice followingtransfer of 2 μl of pooled sera from CD46-IFNAR mice immunized once with10⁶ TCID₅₀ of MVSchw-sE_(WNV). As little as 1 μl of this antiserainduced 66% protection. Passive transfer of sera collected one monthafter a single immunization with 10⁴ TCID₅₀ induced a survival rate of50%. Remarkably, the administration of 1 μl of MV_(Schw)-sE_(WNV)-immunesera collected 1 month after boosting induced 100% protection. Theseresults indicate that a single injection of 10⁶ TCID₅₀ or two injectionsof 10⁴ TCID₅₀ of MV_(Schw)-sE_(WNV) elicited protective humoralresponse. Because the amount of flavivirus inoculated during mosquitofeeding is probably in the order of 10² to 10⁴ infectious virusparticles (1), we assessed the capacity of MV_(Schw)-sE_(WN)-immune serato protect against a range of 10² to 10⁵ focus forming units (FFU) ofWNV strain IS-98-ST1. Groups of six BALB/c mice were passively immunizedwith 2 μl of pooled immune sera collected from CD46-IFNAR mice twiceinoculated with 10⁴ TCID₅₀ of MV_(Schw)-sE_(WNV) (Table 2). Survivalrates of 85-100% were observed in mice that received theMV_(Schw)-sE_(WNV)-immune serum, regardless the lethal doses ofIS-98-ST1 (10 to 10,0001.p. LD₅₀). These data are consistent with thefinding that humoral response plays a critical role in protectionagainst WNV infection.

Mice which are completely unresponsive to IFN-α/β are highly susceptibleto encephalitic flaviviruses (19, 20). Indeed, the inventors previouslyshowed that WNV infection of CD46-IFNAR mice was lethal within 3 daysinstead of 11 days in competent mice (19). To assess whether theimmunity induced by MVSchw-sE_(WNV) could protect these compromisedanimals from WNV infection, three CD46-IFNAR mice from the group thathad received two injections of MV_(Schw)-sE_(WNV) (10⁶ TCID₅₀), werei.p. inoculated with 100 FFU of IS-98-ST1 one month after the boost.Mice inoculated with “empty” MV_(Schw) were used as controls. The micethat had received MV_(Schw)-sE_(WNV) survived the WNV challenge whilecontrol mice died within 3 days. MV_(Schw)-sE_(WNV)-immunized mice werebled 3 weeks after challenge. The FRNT₉₀ antibody response (titer ˜100)was comparable to the pre-challenge response. Notably, post-challengeimmune sera did not react with WNV nonstructural proteins such as NS3and NS5 as shown by RIP assay (FIG. 11, panel MV_(Schw)-sE_(WNV), lane10⁶ TCID₅₀, day 20, p.c.), suggesting that no viral replication occurredafter challenge with WNV. These data show that immunizing withMV_(Schw)-sE_(WNV) prevented WNV infection in highly susceptibleanimals.

The present Example shows for the first time that a live-attenuatedmeasles vector derived from the Schwarz MV vaccine can induce aprotective immunity against an heterologous lethal pathogen. These dataconstitute also the proof of concept that a live-attenuated Schwarzmeasles vaccine engineered to express the secreted form of the WNV Eglycoprotein can be used as a vaccine to prevent West Nile disease inhumans. The MV vaccine vector offers several advantages(over otherexisting viral vectors. The Schwarz MV vaccine has been used on billionsof people since the sixties and shown to be safe and efficacious. It iseasily produced on a large scale in most countries and can bedistributed at low cost. The MV genome is very stable and reversion topathogenicity has never been observed (8). Moreover, MV replicatesexclusively in the cytoplasm, ruling out the possibility of integrationin host DNA. The MV vector has been shown to express a variety of genes,or combinations of genes, of large size over more than twelve passages(6, 16, 21, 22, 23, 24). This stability is likely due to the fact thatthere is little constraint on genome size for pleomorphic viruses with ahelical nucleocapsid. Unlike chimeric viral vectors, the recombinant MVvector is an authentic MV expressing an additional gene. This greatlyreduces the risk, of changing the tropism and the pathogenicity of theoriginal vaccine. It reduces also the risk of recombination.

The recombinant MV-WNV vaccine according to a preferred embodiment ofthe present invention is a promising live-attenuated vector to massimmunize children and adolescents against both measles and West Nilediseases. Although the existence of an anti-MV immunity in nearly theentire adult human population appears to restrict its use to infants, analready worthy goal, recent studies demonstrated that revaccinatingalready immunized children results in a boost of anti-MV antibodies (25,26). These and other studies (Ann Arvin) demonstrated that the presenceof passive MV pre-immunity (maternal antibodies) does not circumvent thereplication of attenuated MV after a second injection. This opens thepossibility of using the live-attenuated MV-derived vector to immunizeadults. Indeed, the inventors reported that a MV-HIV recombinant virusinduced anti-HIV neutralizing antibodies in mice and macaques even inthe presence of pre-existing anti-MV immunity (6). Because ofcross-species transmission, it is feared that WNV becomes a recurrentzoonosis with repeated seasonal outbreaks in humans. The inventorspropose that MVSchw-sE_(WNV) could be used to induce long-term memoryimmunity in large groups of children and adults, and to boost thisimmunity in case of West Nile disease outbreak.

BIBLIOGRAPHY

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TABLE 1 Antibody response of CD46-IFNAR mice to intraperitonealinoculation of MV_(Schw)-sE_(WNV) MV-specific WN-specific WN-specificImmunizing virus Ab titer ⁴ Ab titer ⁴ FRNT₉₀ ⁵ WNV ¹ (10³ FFU) NT10,000 50 MV_(Schw) ² 30,000 <10 <10 (10⁶ TCID₅₀) MV_(Schw)-sE_(WNV) ²15,000 3,000 10 (10⁴ TCID₅₀) MV_(Schw)-sE_(WNV) ² 25,000 3,000 10 (10⁶TCID₅₀) 2 × MV_(Schw)-sE_(WNV) ³ 90,000 30,000 200 (10⁴ TCID₅₀) 2 ×MV_(Schw)-sE_(WNV) ³ 140,000 200,000 300 (10⁶ TCID₅₀) ¹ BALB/c-MBTcongenic mice were i.p. inoculated with WNV strain IS-98-ST1. ² Viruswas given i.p. to CD46-IFNAR mice. ³ Virus was given i.p. twice at 1month of interval. ⁴ Determined by ELISA on pooled heat-inactivatedsera. ⁵ The highest serum dilution that reduced the number of FFU of WNVby at least 90%. NT: not tested

TABLE 2 Protective ability of the MV_(Schw)-sE_(WNV)-immune serum VolumeProtection Material used of sera (no. for transferred ¹ WNV ² surviving/M.D.O.D ³ immunization (□l) (FFU) no. tested) (day ± S.D.) Controls DPBS10 100 0/6 11.5 ± 1.5 WNV ⁴ 10 100 6/6 2 100 5/6 20 MV_(Schw) ⁵ 2 1000/6 12.0 ± 1.5 MV_(Schw)-sE_(WNV) ⁶ 10⁶ TCID50 2 100 6/6 — (day 30) 1100 4/6 11.0 ± 1.5 10⁴ TCID50 10 100 3/6 10.5 ± 2.0 (day 30) 10⁴ TCID501 100 6/6 — (day 60) 2 100 5/6 11 2 1,000 6/6 — 2 10,000 5/6 10 2100,000 5/6 11 ¹ BALB/c mice received 0.1 ml of DPBS containing theindicated amount of pooled sera. ² Mice were challenged with WNV strainIS-98-ST1 one day after passive transfer. ³ Mean day of death ± standarddeviation. ⁴ Immune sera from resistant BALB/c-MBT congenic mice (13)inoculated with 10³ FFU of IS-98-ST1 WNV. ⁵ Immune sera from CD46-IFNARmice collected 30 days after inoculation of MV_(Schw) (10⁶ TCID₅₀). ⁶Immune sera from CD46-IFNAR mice were collected 30 days after 1injection or 60 days after 2 injections of MV_(Schw)-sE_(WNV).

What is claimed is:
 1. A purified polypeptide wherein it derives from aWest-Nile virus antigen or a Dengue virus antigen.
 2. The polypeptideaccording to claim 1, wherein it is capable of inducing a protectiveimmune response against a West-Nile virus or a Dengue virus in ananimal.
 3. The polypeptide according to claim 1 or 2, wherein theWest-Nile virus antigen is selected from the group consisting ofsecreted envelope glycoprotein (E), heterodimer glycoproteins (PreM-E)and NS1 protein.
 4. The polypeptide according to claim 3, wherein thesecreted envelope glycoprotein (E) comprises the sequence of SEQ ID NO:5 or a functional derivative thereof.
 5. The polypeptide according toclaim 3, wherein the heterodimer glycoproteins (PreM-E) comprises thesequence of SEQ ID NO: 6 or a functional derivative thereof.
 6. Thepolypeptide according to claim 3, wherein the NS1 protein comprises thesequence of SEQ ID NO: 7 or a functional derivative thereof.
 7. Thepolypeptide according to claim 1 or 2, wherein the Dengue virus antigenis selected from the group consisting of secreted envelope glycoprotein(E), heterodimer glycoproteins (PreM-E) and NS1 protein.
 8. Thepolypeptide according to claim 7, wherein the heterodimer glycoproteins(PreM-E) comprises the sequence of SEQ ID NO: 8 or a functionalderivative thereof.
 9. The polypeptide according to any one of claims 1to 8, which is an immunogenic peptide.
 10. A purified polyclonal ormonoclonal antibody capable of specifically binding to a polypeptideaccording to any one of claims 1 to 9, or to a fragment thereof.
 11. Anexpression vector comprising a polynucleotide sequence coding for apolypeptide according to any one of claims 1 to
 9. 12. A purifiedpolynucleotide sequence coding for a polypeptide according to any one ofclaims 1 to
 9. 13. The purified polynucleotide sequence of claim 12comprising a sequence selected from the group consisting of SEQ ID Nos:1 to 4 or fragments thereof.
 14. Use of a polynucleotide sequence asdefined in claim 12 or 13 for detecting the presence or absence of aWest-Nile virus antigen or a Dengue virus antigen in a biologicalsample.
 15. A recombinant viral vector which is a recombinant viruscomprising a polynucleotide sequence as defined in claim 12 or
 13. 16.The recombinant viral vector of claim 15, wherein the recombinant virusis a live attenuated virus or a defective virus.
 17. The recombinantviral vector of claim 15 or 16, wherein the recombinant virus isselected from the group consisting of measles virus, hepatitis B virus,human papillomavirus, picornaviridae and lentivirus.
 18. A recombinantmeasles virus capable of expressing a polypeptide according to any oneof claim 1 to
 9. 19. A recombinant measles virus comprising, in itsgenome, a polynucleotide according to claim 12 or
 13. 20. Therecombinant measles virus of claim 18 or 19, which is a live attenuatedvirus or a defective virus.
 21. The recombinant measles virus accordingto any one of claims 18 to 20, which is derived from the Schwarz measlesvirus strain.
 22. A pharmaceutical composition comprising: a) at leastone component selected from the group consisting of: a polypeptideaccording to any one of claims 1 to 9 or a functional derivativethereof; an antibody according to claim 10; an expression vectoraccording to claim 11; a polynucleotide according to claim 12 or 13 or afragment thereof; a recombinant viral vector according to any one ofclaims 15 to 17; and a recombinant measles virus according to any one ofclaims 18 to 21; and b) a pharmaceutically acceptable vehicle orcarrier.
 23. The pharmaceutical composition of claim 22, capable ofinducing a protective immunity against a West-Nile virus or a Denguevirus in an animal.
 24. Use of a pharmaceutical composition according toclaim 22, as an anti-West-Nile virus agent, or for the preparation of ananti-West-Nile virus vaccine.
 25. Use of a pharmaceutical compositionaccording to claim 22, as an anti-Dengue virus agent, or for thepreparation of an anti-Dengue virus vaccine.
 26. A host cellincorporating an expression vector as defined in claim 11 or arecombinant viral vector as defined in any one of claims 15 to
 17. 27.Method of producing a recombinant virus for the preparation of ananti-West-Nile virus vaccine or an anti-Dengue virus vaccine, the methodcomprising the steps of a) providing a host cell as defined in claim 26;b) placing the host cell from step a) in conditions permitting thereplication of a recombinant virus capable of expressing a polypeptideaccording to any one of claims 1 to 9; and c) isolating the recombinantvirus produced in step b).
 28. The cell line deposited at the C.N.C.M.under accession number I-3018.
 29. A West-Nile virus neutralizationassay, comprising the steps of: a) contacting VERO cells with West-Nilevirus and an antibody; b) culturing said VERO cells under conditionswhich allow for West-Nile virus replication; and c) measuring reductionof West-Nile virus replication foci on said VERO cells.
 30. A method fortreating and/or preventing a WNV- or Dengue virus-associated disease orinfection in an animal, the method comprising the step of administeringto the animal an effective amount of at least one element selected fromthe group consisting of: a polypeptide according to any one of claims 1to 9 or a functional derivative thereof; an antibody according to claim10; an expression vector according to claim 11; a polynucleotideaccording to claim 12 or 13 or a fragment thereof, a recombinant viralvector according to any one of claims 15 to 17; and a recombinantmeasles virus according to any one of claims 18 to 21.